Abnormalities of urinary acidification are frequent in chronic renal failure and other kidney diseases. The cellular mechanisms for these disorders are not understood, but likely entail changes in biosynthesis, targetting, and degradation of the kidney H+ ATPase. The object of this proposal is to examine the steps in biosynthesis, and pathways of targetting and degradation of the kidney proton pump in the LLC-PK1 cell line. Cell proteins will be labeled with 35S-methionine under pulse-chase conditions, and immunoprecipitation of pump subunits, using antisera both to the whole enzyme and to individual subunits, will be performed. The analysis will include immunoprecipitation from cytosol, from isolated vacuolar organelle fractions, and from plasma membrane. These studies should allow the precise sequence of assembly of the proton pump and its subsequent intracellular itinerary to be established. These findings will contribute greatly toward elucidating the potential steps from which abnormalities or urinary acidification may arise.